Biological Membranes as Protein Aggregation Matrices and Targets of Amyloid Toxicity - Protein Misfolding and Cellular Stress in Disease and Aging - page 223

in most cases, seem to underlie common biochemical mechanisms
of cytotoxicity ( 2-4 ). One of the leading hypotheses of the molec-
ular basis of amyloid toxicity claims that a sub-population of pre-
fibrillar aggregates, assembled into a pore-like fashion, interacts
with cell membranes in the form of toxic annular structures, simi-
lar to those arising from pore-forming toxins, responsible of
membrane permeabilization ( 5, 6 ). Therefore, the protein-lipid
environment seems to be a physico-chemical feature playing a
crucial role on both aggregate growth kinetics and cytotoxicity.

2. Materials

2.1. Atomic Force
Microscopy

1. De-ionized water.
2. Mica substrates.
3. Phosphate-buffered solution, (10 mM KH 2 PO 4 , 150 mM
KCl, pH 6) or another suitable buffer.
4. TappingMode Fluid Cell.
5. Cantilevers (oxide-sharpened silicon nitride tips, Model
DNP-S, works well).
6. Source of filtered (0.2 mm), compressed air or dry N 2 .
7. Optional for cantilever cleaning: UV lamp, high intensity;
Oriel Mod. 6035 pencil-style spectral calibration lamp or
equivalent.
8. Optional: Fluid cell liquid lines (silicone tubing and fittings),
o-ring, clamping devices (for liquid lines), syringes: 1 cc; 5 cc.

2.2. Transmission
Electron Microscopy

1. Uranyl acetate staining: 2.0% (w/v) uranyl acetate (Sigma) in
water (see Notes 1, 2, 4, and 5). The uranyl acetate is supe-
rior to other negative stains such as uranyl formate and phos-
photungstate due to its higher penetrability and minimal
grain size. The stained grids can be stored at room tempera-
ture without adverse effects.
2. Potassium phosphotungstate staining: a 1-3% solution of
phosphotungstic acid (Sigma) in water; pH is adjusted to
7-7.3 using sodium hydroxide (see Notes 1, 3, and 4).
3. Dilution buffer: 20 mM Tris-HCl, pH 7.5, 200 mM KCl,
1.0 mM DTT or another suitable buffer.
4. Formvar/carbon-coated 400 mesh nickel grids (Agar
Scientific, Stansted, UK).

2.3. Confocal
Immunofluorescence
and Flow Cytometric
Analyses

1. Culture medium: Dulbecco's Modified Eagle's Medium
(DMEM)/F-12 Ham (in a 1:1 ratio), 25 mM N-2-
hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES)
and NaHCO 3 supplemented with 10% fetal bovine serum